Fig. 4. NRF2 was a critical effector of CREB1-mediated GSH dynamics.

(A to C) Real-time qPCR (n = 6) (A), Western blot (B), and immunostaining (C) assays for NRF2 expression (green) in FSK-primed hES-MSCs in the absence or presence of CREB1 silencing (shCREB1) or expression of CREB1-DN inhibitor. The expression level of the indicated proteins was normalized with that of β-ACTIN, which was used as a loading control (n = 3) [(B), right]. (C) Representative images for confocal microscopy are shown with ×1000 magnification. Scale bars, 10 μm. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). (D) FR plots of hES-MSCs carrying control (shCTR) or two independent NRF2 shRNA (shNRF2) constructs (n = 6). (E and F) Real-time qPCR assays evaluating the induction (E) or recruitment of NRF2 (F) in the promoter regions of the indicated NRF2 target genes by FSK priming (n = 4). (G and H) Real-time qPCR assay of NRF2 target genes in FSK-primed hES-MSCs in the absence or presence of NRF2 (G) or CREB1 (H) silencing (n = 4). (I and J) Real-time qPCR (n = 4) (I) and Western blot (J) assays for the expression of CREB1-NRF2–dependent GSH synthesis (GCLM and GCLC) and redox cycling (GSR and PRDX1) genes. The expression level of the indicated proteins was normalized with that of β-ACTIN (n = 3) [(J), right]. *Nonspecific band. (K) FR plot in NRF2 silenced (shNRF2_#1) hES-MSCs, which were rescued by ectopic expression of the indicated CREB1-NRF2 target genes (n = 6). GI values are depicted in the right panel of the FR plot. Data are presented as ratios relative to the value in the control groups and are expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 relative to NT or control group. Statistical analyses were performed using nonparametric Mann-Whitney U test (A), one-way ANOVA (E and F), or two-way ANOVA (B, D, and G to K) with Bonferroni post hoc tests.